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First published online December 3, 2008
doi: 10.1242/10.1242/jcs.036038

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Different GPI-attachment signals affect the oligomerisation of GPI-anchored proteins and their apical sorting

¹ Dipartimento di Biologia e Patologia Cellulare e Molecolare, Università degli Studi di Napoli Federico II
² CEINGE Biotecnologie Avanzate Scarl, via S. Pansini 5, 80131 Napoli, Italy
³ Unité de Trafic Membranaire et Pathogénèse, Institut Pasteur, 25 rue du Docteur Roux, Paris 75724, France

View larger version (24K): [in this window] [in a new window]   Fig. 1. GFP-fusion proteins and their treatment with PI-PLC. (A) In the fusion constructs GFP-FR and GFP-PrP, GFP is fused to the GPI-anchor attachment signal of folate receptor (FR) and prion protein (PrP), respectively. The FR or PrP sequence included in the fusion proteins is shown. The -site is shown in red. (B) MDCK cells stably expressing GFP-FR or GFP-PrP were extracted with Triton X-114. After phase separation, detergent phases were incubated in the presence (+) or absence (–) of PI-PLC (15 µg/ml) as previously described (Lisanti et al., 1989). The resulting aqueous phases were precipitated with trichloroacetic acid and detected by western blotting using anti-GFP antibody. As previously shown, the band migrating at 43 kDa represents a partially denatured dimer of GFP (Paladino et al., 2004).

View larger version (33K): [in this window] [in a new window]   Fig. 2. GFP-FR and GFP-PrP are differently sorted in MDCK cells. MDCK cells stably expressing GFP-FR or GFP-PrP were grown on filter for 4 days. (A,B) Cells were analysed in vivo in (A) CO2-independent medium or (B) stained with an anti-GFP antibody followed by a TRITC-conjugated secondary antibody under non-permeabilising conditions. Serial confocal sections were collected from top to bottom of cell monolayers. (C) Cells were labelled with LC-biotin, which was added to their apical (Ap) or basolateral (Bl) surface. After immunoprecipitation using anti-GFP antibody, samples were separated by SDS-PAGE and biotinylated proteins were revealed using HRP-streptavidin. Bars, 10 µm.

View larger version (25K): [in this window] [in a new window]   Fig. 3. GFP-FR and GFP-PrP both associate with DRMs, but only GFP-FR forms HMM complexes. (A) MDCK cells stably expressing GFP-FR or GFP-PrP were lysed at 4°C in buffer containing 1% Triton X-100 and separated by centrifugation until equilibrium on 5-40% sucrose-density gradients to purify Triton-X-100 insoluble microdomains. Fractions of 1 ml were collected from top (fraction 1) to bottom (fraction 12) and, after TCA-precipitation, run on SDS-PAGE and detected using anti-GFP antibody. One aliquot of each fraction was transferred onto nitrocellulose membrane, and GM1 (a typical raft-marker) was revealed by using cholera toxin conjugated to HRP. (B) Cells were lysed in buffer containing 0.4% SDS and 0.2% Triton X-100 and run through 5-30% sucrose gradients. Fractions of 500 µl were collected from the top (fraction 1) to the bottom (fraction 9) of the gradients. Proteins were TCA-precipitated and detected by western blotting using a specific GFP antibody. The position on the gradients of molecular mass markers is indicated. The graphs show the mean values of protein distribution on the gradients from three different experiments ± s.d.

View larger version (41K): [in this window] [in a new window]   Fig. 4. Addition of cholesterol affects the oligomeric state and the polarity of GFP-PrP but does not influence the behaviour of GFP-FR. MDCK cells stably expressing GFP-FR or GFP-PrP were loaded with 2 mM cholesterol (+chol) or not (control), and oligomerisation state and distribution of GFP-FR and GFP-PrP at the plasma membrane were assessed. (A) Cells were lysed as decribed for Fig. 3B and ran through 20-40% glycerol gradients. Fractions of 300 µl were collected from the top (fraction 2) to the bottom (fraction 15) of the gradients. Proteins were precipitated with TCA and detected by western blotting using anti-GFP antibody. The position on the gradients of the molecular mass markers is indicated. (B,C) Plasma membrane localisation was determined by analysing (B) the natural fluorescence of GFP or by (C) an immunofluorescence assay performed under non-permeabilising conditions by adding anti-GFP antibody to the apical side of cells that had been grown on filter for 4 days. Serial confocal sections were collected from the top to the bottom of the cells. Mean fluorescence intensities at the apical and basolateral domains were measured and are expressed as percentages of total fluorescence. Bars, 15 µm.

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